Enteric Pathology and Salmonella-Induced

Cell Death in Healthy and SIV-Infected

Rhesus Macaques

Veterinary Pathology
48(5) 933-941
The American College of
Veterinary Pathologists 2011
Reprints and permission:
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DOI: 10.1177/0300985810386468
http://vet.sagepub.com

R. L. Santos1, A. P. Almeida1, M. N. Xavier1, T. A. Paixao1,

R. P. Wilson2, S. Dandekar2, M. Raffatellu2, and A. J. Baumler2

Abstract
The goal of this study was to morphologically characterize a ligated ileal loop model of Salmonella enterica serotype Typhimurium
infection in rhesus macaques (Macaca mulatta) and to verify the occurrence of Salmonella-induced cell death in vivo. Eight adult
healthy male rhesus macaques were used for ligated ileal loop surgery. Four macaques had been intravenously inoculated with
simian immunodeficiency virus (SIV) mac251. Ileal ligated loops were inoculated with wild-type (WT) S. Typhimurium strain
IR715 (ATCC14028 nalr), an isogenic noninvasive mutant strain (ATCC14028 nalr DsipADsopABDE2), or sterile Luria Bertani
broth. Loops were surgically removed at 2, 5, and 8 hours post-inoculation (hpi). Intestinal samples were processed for
histopathology, immunohistochemistry for detecting Salmonella, terminal deoxynucleotidyl transferase-mediated dUTP-biotin
nick end labeling (TUNEL), and transmission electron microscopy. Combined histopathology scores were similar between
SIV-infected and control macaques. As expected, the invasion-deficient mutant was less pathogenic than WT S. Typhimurium.
Neutrophil infiltrate in the intestinal mucosa correlated with bacterial loads (r 0.7148; P < .0001) and fluid accumulation
(r 0.6019; P < .0001) in the lumen of the intestinal loops. Immunolabeled WT S. Typhimurium was observed in the epithelium
and lamina propria at the tip of the villi at 2 hpi, progressing toward deeper lamina propria at 58 hpi. Most TUNEL-positive cells
localized to the lamina propria, and some had morphological features of macrophages. Ultrastructurally, bacteria were observed
intracellularly in the lamina propria as well as within apoptotic bodies. This study provides morphological evidence of Salmonellainduced cell death in vivo in a relevant nonhuman primate model.
Keywords
apoptosis, inflammation, macrophages, rhesus monkeys, Salmonella, simian immunodeficiency virus

Nontyphoidal Salmonella serotypes (NTSs) are a major cause

of food-borne infections worldwide. NTS infection is primarily
characterized by enterocolitis and only rarely results in bacteremia.23 Once in the intestinal lumen, NTSs readily invade the
intestinal epithelium, including M cells and enterocytes,
quickly reaching the lamina propria, where they are internalized by phagocytes.22 During this invasive process, NTSs trigger a strong acute inflammatory reaction resulting in extensive
tissue damage.24 It has been demonstrated that the invasive
phenotype of NTS is dependent on 5 effector proteins secreted
through the type III secretion system encoded by the Salmonella
pathogenicity island 1 (SPI-1).35 Although systemic dissemination of NTS or bacteremia is a rare complication in immunocompetent individuals, it occurs commonly among patients with
human immunodeficiency virus (HIV) infection, resulting in
up to 47% mortality.10 We have recently demonstrated that SIV
infection in rhesus macaques, a model for HIV infection
in humans, promotes systemic dissemination of Salmonella
enterica serotype Typhimurium (S. Typhimurium) after intestinal invasion.19

Early studies have demonstrated that S. Typhimurium

induced macrophage death requires a functional SPI-1 encoded
type III secretion system.13,14,25 Recently, it has been demonstrated that flagellin secreted through the SPI-1 encoded type
III secretion system is responsible for triggering cell death
by acting as an agonist of the intracellular nucleotide-binding
and oligomerization domain (NOD)like receptor Ipaf, leading
to activation of caspase-1.7,15 Since interleukin (IL)-1b
and IL-18 are cleaved and activated by caspase 1, this

Departamento de Clinica e Cirurgia Veterinaria, Escola de Veterinaria da

Universidade Federal de Minas Gerais, Belo Horizonte, MG, Brazil
2
Department of Medical Microbiology and Immunology, University of
California at Davis, Davis, CA
Corresponding Author:
Renato L. Santos, DVM, MS, PhD, Departamento de Clinica e Cirurgia
Veterinaria, Escola de Veterinaria da Universidade Federal de Minas Gerais,
Av. Antonio Carlos, 6627, Belo Horizonte, MG, Brazil
Email: rsantos@vet.ufmg.br

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Table 1. SIV Infection Status of Individual Macaques Used in this Study

a

Animal

Group

SIV Infection

1
2
3
4
5
6
7
8

SIV
SIV
CONTROL
SIV
CONTROL
CONTROL
CONTROL
SIV

43 days
50 days
Not applicable
71 days
Not applicable
Not applicable
Not applicable
71 days

Duration between SIV inoculation and Salmonella enterica serotype typhimurium

infection.

Salmonella-induced macrophage death is considered a

proinflammatory mechanism, and therefore it has been
designated pyroptosis.2 Although several studies have
addressed the molecular mechanisms of Salmonella-induced
macrophage death in vitro,7,15 its significance in a relevant
animal model remains to be demonstrated. A previous study
on S. Typhimurium infection of bovine ligated ileal loops did
not report an increase in Salmonella-induced cell death26 in this
animal model of NTS-induced enterocolitis.28
The goal of this study was to morphologically characterize a
ligated ileal loop model of S. Typhimurium infection in rhesus
macaques and investigate the occurrence of Salmonellainduced cell death in vivo.

Material and Methods

Animals and Experimental Design

At 510 weeks after SIV inoculation (Table 1), SIV-infected

macaques were anesthetized with ketamine (10 mg/kg
intramuscularly; Parke-Davis), followed by placement of an
endotracheal tube and maintenance of anesthesia with isoflurane (12%). When needed, ventilation was maintained with a
positive-pressure respirator. Vital signs including heart rate,
respiratory rate, and blood pressure were monitored. A laparotomy was performed, exposing the ileum and ligating 13 loops
with an average of 4 cm in length, leaving 1-cm spacer loops in
between. The loops were inoculated by intraluminal injections
of 1 ml of either sterile Luria Bertani (LB) broth, a logarithmically grown culture containing 1  109 colony-forming units
(CFUs) of wild-type S. Typhimurium (IR715),19 or an isogenic
invasion-deficient mutant (sipA sopA sopB sopD sopE2
mutant) of S. Typhimurium (strain ZA21).35 Loops were
surgically removed at 2, 5, or 8 hours after inoculation.19,35 An
additional 4 loops were inoculated with another S. Typhimurium
strain that was not included in this study. Samples for bacteriology
were collected from the antimesenteric side of the intestinal
mucosa using 6-mm biopsy punches. Fragments of the intestinal
mucosa were incubated for 1 hour in phosphate-buffered saline
(PBS) containing gentamicin at 50 mg/1 (to inactivate extracellular bacteria), homogenized, serially diluted, and plated on LB agar
plates containing the appropriate antibiotics. The volume of fluid
in each loop was estimated by the difference in the weight of loops
before and after draining all the luminal fluid contents. None of
the SIV-infected macaques developed clinical signs associated
with immunodeficiency prior to surgery.

Histopathology and Immunohistochemistry

All experiments were approved by the Institutional Animal

Care and Use Committee at the University of California, Davis.
Eight healthy, Salmonella-free (as determined by fecal culture), 2- to 4-year-old male rhesus macaques from a colony
known to be free of SIV were used for ligated ileal loop surgery
in this study (Table 1). The animals were kept in individual
cages, receiving water ad libitum and commercial monkey
chow. Four randomly selected macaques (macaque Nos. 1, 2,
4, and 8) were intravenously inoculated with 1000 TCID50
(tissue culture infective dose) previously titrated frozen stocks
of SIV mac251 grown on peripheral blood mononuclear cells.
Plasma viral loads were determined as previously
described.9 Briefly, plasma samples were subjected to quantitative reverse transcriptionpolymerase chain reaction (RT-PCR)
analyses to determine the level of SIV infection. Primers
and probes specific to the SIV RNA sequence were used.
Probes were tagged with a fluorescent reporter dye at the 50 end
and a quencher dye at the 30 end. Fluorescence signal was
detected with an ABI Prism 7700 sequence detector (PE
Applied Biosystems). Data were captured and analyzed with
Sequence Detector Software. Viral copy number was determined by plotting computed tomographic values obtained from
the plasma samples against a standard curve derived from
samples with known viral copy numbers. 9

Fragments from the Peyers patches were fixed by immersion

in 10% buffered formalin for 2448 hours, embedded in
paraffin, sectioned at 5-mm thickness, and stained with hematoxylin and eosin. Neutrophil infiltration was scored from
0 to 3 according to the following criteria: 0, no infiltration; 1,
mild diffuse infiltration of neutrophils at the tips of absorptive
villi; 2, moderate diffuse infiltration of neutrophils in
the mucosa; and 3, severe diffuse infiltration of neutrophils
in the mucosa and mild to moderate infiltration in the submucosa. Similarly, other histopathological changes were scored,
including hemorrhage (0, absence of hemorrhage; 1, mild focal
hemorrhage; 2, moderate multifocal hemorrhage; 3, diffuse
hemorrhage), cell death (necrosis and/or apoptosis), for which
the scoring system was based on the number of cells with
morphological features of cell death, particularly chromatin
condensation and fragmentation (0, absence of cells with morphological features of cell death; 1, a few necrotic/apoptotic
cells; 2, moderate numbers of apoptotic/necrotic cells; 3, large
numbers of apoptotic/necrotic cells in all villi), blunting of villi
(0, normal villi; 1, some of the villi mildly blunted; 2, most of
the villi moderately blunted; 3, all villi blunted with most villi
severely blunted), epithelial loss (0, absence of epithelial loss;
1, detachment of a few enterocytes at the tip of a few villi; 2,
detachment of some enterocytes in some villi; 3, detachment
of several enterocytes at the tip of most villi), and edema

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935
deparaffinized, hydrated, and treated with a proteinase K (Sigma,
St. Louis, MO) solution (20 mg/ml) and 0.5% Triton X-100
(Sigma). In situ detection of cells undergoing cell death was
performed using a commercial kit (Apoptag Plus kit; Intergen,
Purchase, NY) according to the manufacturers instructions.
Sections of normal female rodent mammary gland, obtained
35 days after weaning of rat pups, were used as positive controls.
Negative controls were obtained by replacing the TdT with buffer
on the same tissues used as positive control. Scores were attributed
to the intensity of TUNEL staining as follows: absence of staining
0; mild 1; moderate 2; and intense 3.

Transmission Electron Microscopy

Figure 1. Plasma viral loads of SIV-infected rhesus macaques, animal
Nos. 1, 2, 4, and 8, based on quantitative RT-PCR as determined in (19).

(0, absence of edema; 1, mild edema characterized by mild

and/or focal accumulation of interstitial fluid; 2, moderate
edema with moderate and/or multifocal to coalescent accumulation of interstitial fluid with separation of collagen fibers and
dilation of lymphatic vessels; 3, severe edema characterized by
diffuse and severe accumulation of interstitial fluid with marked
separation of collagen fibers and dilation of lymphatic vessels).
All slides were coded and evaluated by a veterinary pathologist
(R. L. S.) without knowledge of their infection status.
Immunohistochemistry was performed as previously
described.21 Serotype Typhimurium was detected in tissue
sections by using a purified polyclonal antibody reactive to
Salmonella lipopolysaccharide (Biodesign International, Saco,
ME). A commercial avidinbiotinperoxidase kit (LSAB1 Kit,
DAKO Corporation, Carpinteria, CA) was used as detection
system according to the manufacturers instructions. Briefly,
5-mm sections were hydrated and incubated in 4% hydrogen peroxide in PBS (0.01 M, pH 7.2), incubated with skim milk (2.6% in
distilled water) as blocking antibody, and then incubated with the
primary antibody (1:10,000 dilution) for 1516 hours at 40 C in a
humid chamber. After washing in PBS, the slides were incubated
with biotinylated secondary antibody for 20 minutes at room temperature, washed in PBS again, and then incubated with streptavidinperoxidase complex for 20 minutes at room temperature. The
reaction was developed with a 0.024% diaminobenzidine (Sigma,
St. Louis, MO) solution and 0.16% hydrogen peroxide. The slides
were counterstained with Harris hematoxylin. For negative
controls, we used tissues known to be free of Salmonella with the
primary antibody replaced by PBS. Tissue distribution of
immunolabeled S. Typhimurium in different components of the
intestinal mucosa and submucosa was qualitatively determined
(ie, presence or absence of organisms, regardless of the number
or intensity of staining).

TUNEL Staining
A TUNEL assay8 was used for in situ detection of cells undergoing
cell death. Five-micrometer sections of the Peyers patches were

Small fragments of intestinal mucosa were fixed by immersion

overnight at 4 C in a solution of 5% glutaraldehyde and 4%
paraformaldehyde in 0.1 M sodium cacodylate buffer. Tissues
were then washed 3 times with 0.1 M sodium cacodylate buffer
and postfixed for 2 hours at 4 C in 1% osmium tetroxide in
0.1 M sodium cacodylate buffer. For transmission electron
microscopy, the samples were stained overnight at 4 C in a
saturated uranyl acetate solution. Tissues were dehydrated in
a graduated series of ethanol solutions and propylene oxide and
embedded in Epon Araldite. Sections (0.5 mm) were stained
with toluidine blue and examined under a light microscope for
selection of the microscopic fields. The blocks were trimmed,
and thin sections (6090 nm) were cut, mounted onto copper
grids, stained with uranyl acetate and lead citrate, and examined with a Philips CM120 Biotwin Lens transmission electron
microscope (Eindhoven, The Netherlands).

Statistical Analysis
Semiquantitative data were analyzed by the nonparametric
method of Kruskal-Wallis. CFU numbers underwent logarithmic
transformation prior to analysis of variance (ANOVA) and mean
comparisons using the Tukey test. Selected sets of data were subjected to Pearsons correlation analysis. Data are presented as
mean + standard error of the mean. P values were considered
significant when P < .05. Statistical analyses were performed
using the Graphpad Instat software 3.05 (Graphpad Software,
Inc., San Diego, CA).

Results
S. Typhimurium-Induced Intestinal Histopathology
All animals inoculated with SIV became infected as
demonstrated by quantitative RT-PCR. Plasma viral loads
of SIV-infected animals are demonstrated in Fig. 1.
Histopathology scores for hemorrhage, cell death, blunting
of villi, epithelial loss, edema, and neutrophil infiltration are
presented in Fig. 2. The combined histopathology scores were
similar between SIV-infected and control macaques throughout
the time course of infection (Fig. 2). As expected, the invasiondeficient mutant (strain ZA21) was less pathogenic (ie, induced
a lower combined histopathology score) compared with the

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Figure 2. Combined histopathology scores. The scores for histologic parameters (0 for absent or 13 for mild to severe, respectively) were
added, and the data shown are the average combined score from the 4 macaques in each group. Macaques were SIV-infected (n 4) or control
(n 4). Intestinal loops were inoculated with sterile broth (LB), wild-type S. Typhimurium (WT), or the noninvasive mutant of S. Typhimurium
(ZA21). Combined scores were compared by the Kruskal-Wallis test, and statistically significant differences are indicated (P < .05).

wild-type S. Typhimurium (strain IR715) (Fig. 2). A marked

diffuse neutrophilic infiltration was observed at 5 and 8 hours
post-inoculation (hpi), which was associated with other
secondary inflammatory changes such as edema and blunting
of villi (Figs. 3, 4). Histopathological changes, particularly
neutrophil infiltration, correlated well with bacterial loads
(correlation of score of neutrophil infiltration and log of
CFU numbers: r 0.7148; P < .0001) and fluid accumulation
(r 0.6019; P < .0001) in the lumen of the intestinal loops.
Bacterial numbers were consistently higher in the intestinal
mucosa of loops infected with wild-type S. Typhimurium
(strain IR715) compared with the mucosa of loops inoculated
with the invasion-deficient mutant (strain ZA21), in both
control and SIV-infected macaques (Fig. 5). Fluid accumulation increased over the time course of infection, with larger
volumes of fluid accumulating in loops infected with the
wild-type strain (strain IR715) compared with loops infected
with the invasion-deficient mutant (strain ZA21). Loops
inoculated with sterile LB broth accumulated no fluid.
SIV-infected macaques tended to accumulate more fluid in
response to S. Typhimurium infection than nonSIV-infected
macaques, but this difference was not significant (Fig. 6).

Tissue Distribution of S. Typhimurium

The kinetics of tissue invasion were evaluated by immunohistochemical labeling of S. Typhimurium. Immunolabeled
wild-type S. Typhimurium was observed in the epithelium and
lamina propria at the tip of the villi at 2 hpi, progressing toward
deeper lamina propria at 58 hpi and reaching the submucosa
and lymphoid follicles at 8 hpi in animal No. 5 (Table 2),
in which immunolabeled S. Typhimurium were observed in
lymphoid follicles at earlier time points. Similar kinetics of
invasion were observed in SIV-infected macaques, although
the number of animals with immunolabeled S. Typhimurium
tended to be higher in deeper tissues in these animals compared
with control macaques (Table 2). As expected, the invasiondeficient mutant (strain ZA21) remained mostly restricted to
the lumen and apical epithelial surface throughout the time
course of the study (Table 2, Figs. 7, 8).

S. Typhimurium-Induced Cell Death

In this study, cells in the intestinal mucosa were labeled by the
TUNEL technique, and morphological features of apoptotic

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937

Figure 3. Small intestine; rhesus macaque, animal No. 6 (not infected

with SIV) at 5 hours post-infection of the intestinal loop with sterile LB
broth (no infection of the intestinal loop with S. Typhimurium). Intestinal
villus with no significant histological changes. HE. Figure 4. Small intestine; rhesus macaque, animal No. 6 (not infected with SIV) at 5 hours
post-infection of the intestinal loop with wild-type S. Typhimurium (strain
IR715). The intestinal villus is blunted, there is moderate infiltration of
neutrophils and hyperemia, and there is accumulation of apoptotic
bodies and/or necrotic debris in the lamina propria. HE.

cell death were examined by transmission electron microscopy.

Wild-type S. Typhimurium (IR715) infection resulted in an
increase (P < .05) in cell death in the intestinal mucosa as assessed
by TUNEL (Fig. 9). Most of the TUNEL-positive cells localized
to the lamina propria (Figs. 1012). The intensity of TUNEL
labeling was similar between control and SIV-infected macaques.
A significant correlation was observed between the scores for
neutrophil infiltration and TUNEL labeling in the intestinal
mucosa (r 0.7103; P < .0001). Ultrastructurally, bacteria were
observed within the cytosol of macrophages in the lamina propria
as well as within apoptotic bodies (Fig. 13).

Discussion
This is the first in vivo study providing evidence for a direct
interaction of S. Typhimurium with cells undergoing cell death
in a relevant animal model for NTS. Previous studies have
demonstrated the ability of S. Typhimurium to induce
macrophage cell death in vitro.4,5,6,14,17,25,31,32 This effect is
dependent on a functional SPI-1 encoded type III secretion system, and it is mediated by caspase 1. However, it only recently
became clear that activation of caspase 1 is mediated by Ipaf
upon recognition of flagellin that is translocated into the host
cell cytosol through the SPI-1 type III secretion system.7,15,30,31
It has also been recently reported that S. Typhimurium can
induce caspase 1mediated death of stromal cells.18 Considering that the proinflammatory cytokines IL-1 and IL-18 are
substrates of caspase 1, being cleaved and activated during the
caspase 1mediated macrophage cell death, this mechanism of
Salmonella-induced macrophage death is thought to be a
significant proinflammatory mechanism during S. Typhimurium infection, and therefore it has been named pyroptosis,

which can be triggered not only by S. Typhimurium but also

by several infectious or noninfectious pathological stimuli.2
The role of caspase 1 during S. Typhimurium infection has
been studied in the mouse.16,20 However, this animal model has
limitations for studying enterocolitis since mice develop a
systemic disease but not diarrhea in response to S. Typhimurium infection28 and the induction of enterocolitis in mice
requires artificial conditions in which the intestinal flora is
depleted prior to infection.1 Previous attempts to demonstrate
the occurrence of this phenomenon in the calf, a relevant animal model for Salmonella-induced diarrhea, did not provide
evidence for S. Typhimuriuminduced cell death in vivo.26 A
recent study demonstrates that recognition of flagellin by the
host innate immune system contributes to a proinflammatory
response in the calf, which is a suitable model for NTSinduced diarrhea, but not in the mouse.33, 34
Our results support the notion that the presence of bacteria
in proximity to the host target cell is not the only mechanism
responsible for cell death in the intestinal mucosa during
Salmonella infection. This conclusion is supported by the
immunohistochemistry data that have proven to be appropriate
for studying tissue distribution of Salmonella in the intestine.21
This approach demonstrated that an invasion-deficient
S. Typhimurium mutant (strain ZA21)35 induced an increase
in TUNEL-labeled cells in the intestinal mucosa despite the
fact that it remained mostly associated with the epithelial layer
throughout the course of infection, with minimal invasion
of the lamina propria. However, considering the association
of S. Typhimurium with cells undergoing cell death in the
lamina propria and the proinflammatory nature of this mechanism
as demonstrated in cultured macrophages,7,15 it is likely that
Salmonella-induced cell death plays a proinflammatory role in
vivo in this nonhuman primate model.
Nonhuman primates have been used in the past as animal
models for Salmonella infection.12 The adaptation of the
intestinal ligated loop model to rhesus macaques provided a
useful animal model for studying enteric pathogens, resulting
in a significant reduction in the number of animals required for
a given experiment. The pattern of host response observed in
this study characterized by an acute inflammatory response
with abundant neutrophilic infiltration is similar to that previously described in the calf in which the ileal ligated loop model
has been extensively used.26,27
Some of our observations are consistent with the idea that
SIV infection impairs the integrity of the intestinal epithelial
barrier, although the differences between noninfected and
SIV-infected macaques in this study were not statistically significant. First, S. Typhimurium elicited increased fluid accumulation (a surrogate measure of diarrhea) in the intestinal lumen
of SIV-infected compared with control animals. Second,
spreading of wild-type S. Typhimurium into the deeper layers
of the mucosa was more evident in SIV-infected than in control
animals. These observations are in good agreement with previous studies demonstrating that SIV infections result in an
impairment of intestinal mucosal integrity,11 which may favor
bacterial translocation from the intestinal lumen.3 Previous

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Figure 5. Bacterial numbers in intestinal loops of rhesus macaques in response to inoculation with S. Typhimurium. (a) SIV-noninfected (control)
macaques; (b) SIV-infected macaques. Intestinal loops were inoculated with sterile broth (control), wild-type S. Typhimurium (WT), or the noninvasive
mutant of S. Typhimurium (ZA21). CFU numbers that are statistically different from those in control loops not infected with S. Typhimurium
are indicated (**P < .01; ***P < .001; ANOVA with Tukeys comparison of means).

Figure 6. Fluid accumulation in intestinal loops of rhesus macaques in response to inoculation with S. Typhimurium. (a) SIV-noninfected (control)
macaques; (b) SIV-infected macaques. Intestinal loops were inoculated with sterile broth (control), wild-type S. Typhimurium (WT), or the
noninvasive mutant of S. Typhimurium (ZA21). Statistically significant differences are indicated (ANOVA with Tukeys comparison of means).
Table 2. Summary of Immunolabeling of Salmonella enterica Serotype Typhimurium in Ligated Intestinal Loops of Uninfected Controls or
SIV-Infected Rhesus Macaques Inoculated With the Wild-Type Strain or an Invasion-Defective Mutant (ZA21)
Uninfected Controls
WT

L
Ep
LPt
LPm
LPb
SM
LF

SIV-Infected Animals
ZA21

WT

ZA21

2 hpi

5 hpi

8 hpi

2 hpi

5 hpi

8 hpi

2 hpi

5 hpi

8 hpi

2 hpi

5 hpi

8 hpi

E, epithelium; hpi, hours post-inoculation; L, intestinal lumen; LF, lymphoid follicle; LPb, lamina propria of the base of the villi; LPm, lamina propria of the middle of the villi;
LPt, lamina propria of the tip of the villi; SM, submucosa; WT, wild-type. The number of marks corresponds to the number of positive macaques (n 4 per group).
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Figure 7. Small intestine; rhesus macaque, animal No. 7 (not infected

with SIV) at 8 hours post-infection of the intestinal loop with wild-type
S. Typhimurium (strain IR715). Intestinal villus with intense and diffuse
immunolabeling of Salmonella sp. in the lamina propria. Polyclonal
antibody against S. Typhimurium lipopolysaccharide (LPS), counterstained with hematoxylin. Figure 8. Small intestine; rhesus macaque,
animal No. 3 (not infected with SIV) at 8 hours post-infection of the
intestinal loop with the noninvasive mutant of S. Typhimurium (strain
ZA21). Intestinal villus with rare immunolabeled Salmonella sp. in the
lamina propria and abundant immunolabeled organisms in the intestinal
lumen. Polyclonal antibody against S. Typhimurium LPS, counterstained
with hematoxylin.

Figure 1012. Small intestine; rhesus macaque, animal No. 3 (not

infected with SIV) at 8 hours post-infection of the intestinal loop with
sterile LB broth (uninfected control loop; Figure 10), with the
noninvasive mutant (strain ZA21) of S. Typhimurium (Figure 11), or with
wild-type (strain IR715) S. Typhimurium (Figure 12). Intestinal villus with
a few TUNEL-positive cells in the lamina propria (Figures 10, 11) or
with large numbers of diffusely distributed TUNEL-positive cells in the
lamina propria (Figure 12). Terminal transferase dUTP nick end labeling
(TUNEL), counterstained with methyl green.

Figure 13. Small intestine, lamina propria; rhesus macaque, animal

No. 1 (SIV-infected macaque) at 5 hours post-infection of the intestinal
loop with wild-type S. Typhimurium (strain IR715). Bacterial organisms
with morphology compatible with Salmonella sp., characterized by
multiple bacilli within an apoptotic body in the lamina propria (arrow).
Transmission electron microscopy.

Figure 9. Average scores of TUNEL staining in the intestinal mucosa

of loops from control (a, n 4) or SIV-infected (b, n 4) rhesus
macaques inoculated with S. Typhimurium, either wild-type (strain
IR715) or the noninvasive mutant (strain ZA21), and uninfected
controls. There were no statistically significant differences (P > .05).

work suggests that the mechanism for SIV-mediated defects

of mucosal barrier function is a depletion of TH17 cells.19
SIV-induced changes in intestinal barrier and absorption are not
associated with intestinal bacterial overgrowth,29 which could
interfere with the experimental model employed in this study.
In conclusion, here we describe the intestinal host response
to S. Typhimurium. This study also provides the first in vivo
evidence of the association of S. Typhimurium with cells undergoing cell death in the intestinal mucosa in a relevant biological
model.

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Acknowledgements
We thank G. Adamson (Electron Microscopy Laboratory, Department
of Medical Pathology and Laboratory MedicineUC Davis) for
technical assistance.

11.

Declaration of Conflicting Interests

The authors declared that they had no conflicts of interest with respect
to their authorship or the publication of this article.

12.

Financial Disclosure/Funding

13.

Work in RLSs laboratory is supported by CNPq (Conselho Nacional

de Desenvolvimento Cientfico e Tecnologico, Brazil) and FAPEMIG
(Fundacao de Amparo a Pesquisa do Estado de Minas Gerais, Brazil).
RLS, TAP, and MNX are recipients of fellowships from CNPq
(Brazil). RLS is a fellow of the John Simon Guggenheim Memorial
Foundation. Work in AJBs laboratory is supported by Public Health
Service Grants AI040124, AI044170, AI073120, AI076246 and
AI079173.

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